Species Identification And Postmortem Interval Estimation Based On The Cuticular Hydrocarbons Of Necrophagous Flies

Background: Entomological evidence can provide a particular perspective and thinking direction for forensic medicine,especially in solving the Postmortem interval(PMI)inference,and other aspects of death can provide a breakthrough point and clues for case detection.PMI inference is mainly based on morphological characteristics and growth and development rules of necrophilic flies.However,there are significant differences in the development time of different species,so species identification is still a crucial link in practical case applications.There are still some deficiencies in species identification,such as it is challenging to distinguish closely related species accurately,and the development time is mainly inferred by the morphological changes of larvae and live pupae on corpses.However,there is still no effective method to infer the death cases with only the empty puparium.On the other hand,the cuticle hydrocarbons of flies are widely present in various insect stages and empty puparium.They are characterized by speciation-specificity,time-series expression difference,and high stability,which is expected to make up for the shortcomings of existing inference methods and provide a more scientific and complete method system for PMI inference using necrophilic flies.Objective:(1)To study the different profiles of CHCs in the different necrophilic flies and analyze their potential value in species identification;(2)To study the changes of CHCs in different ages of Sarcophagus peregrina and to improve the development time inference method;(3)To study the weathering of empty puparium of Sarcophagus peregrina under different temperatures.Methods:(1)Pig lung was used to attract necrophilic flies in Changsha,Hunan Province.After morphological species identification,the profiles of CHCs of each fly were detected by GC-MS,and the OPLS-DA model was constructed to analyze and compare the composition differences of different species;(2)Sarcophagus peregrina was chosen as the study fly.The adults were reared in the incubator with the condition of 25℃,75%HD,12D:12L,and then pig lung was used to induce larviposition.The offspring were reared in the same condition;Samples were collected in all stages and using the OPLS-DA model to compare the difference of CHCs.Compounds with noticeable differences were selected to construct fitting equations for age inference;(3)The empty pupariums of Sarcophagus peregrina were put in three incubators under 10℃,25℃,40℃,while other conditions were set as before.The samples were collected for 120 days to compare the hydrocarbon weathering tendency at different temperatures.PLS and SVR regression models were constructed to predict the weathering time of empty puparium at different temperatures.Results:(1)We collected 13 species of Sarcophagidae,12 species of Calliphoridae,and two species of Muscidae.GC-MS identified 102 types of CHCs.These three families could be distinguished using OPLS-DA,especially for Sarcophaga peregrina vs.Sarcophaga formosensis.(2)In the whole stage of Sarcophaga peregrina,63 types of CHCs between C8 and C36 were identified by GC-MS.N-alkanes and branch alkanes were the two primary compounds of all detected hydrocarbons,while branch alkanes dominated with time.High molecular weight hydrocarbons,as 2-Methyl C19,Di Methyl C21,C22,and C23,existed the whole life cycle and were temporality.(3)We collected the puparium of Sarcophaga peregrina in 120 days at three temperatures(10℃,25℃,40℃)and identified 49 CHCs with the carbon chain length between 10 and 33 by GC-MS.The abundance of the hydrocarbons decreased with weathering time significantly at a low temperature while it escalated with time at high and moderate temperatures.PLS and SVR models show that the variation tendency of hydrocarbon could be used to estimate the weathering time.The R2 of SVR exceeds 84.2%.Conclusion:(1)The species differences of hydrocarbon compositions in the cuticle of adult necrophilic flies were explored,the differences of CHCs of relatives were revealed,which made up for the deficiency of PMI in identification of relatives species.;(2)The difference in hydrocarbon composition in the epidermis of S.peregrina at different ages was compared and analyzed.The changing pattern of hydrocarbon composition in larva,pupal,and adult was discussed,which expanded the method of development time inference and improved the accuracy of existing PMI inference.(3)The influence of temperature on the empty puparium weathering rate of S.peregrina was explored,and the empty puparium weathering time inference method was improved,providing a research direction for the long-term PMI inference.